The expression of the mitochondrial uncoupling proteins (UCP) 2 and UCPS in the heart has been shown to change in response to a variety of metabolic stressors, including pressure overload and congestive heart failure. Furthermore, the overexpression of UCPs in isolated myocytes has been shown to increase fatty acid oxidation and to improve myocyte survival in the setting of hypoxia. UCPs are believed to serve several purposes: (1) uncouple citric acid cycle flux from mitochondrial ATP synthesis, (2) facilitate fatty acid metabolism and (3) decrease reactive oxygen species production. However, the contribution of UCPs to each of these functions in the intact, beating heart and the protective role of UCPs in the intact heart are not known. We hypothesize that increased expression of UCPs in the heart is associated with increased complete oxidation of fatty acids with decreased generation of lipid-derived free radicals, despite changes in the coupling of citric acid cycle flux and ATP generation, and that increased expression of UCPs is cardioprotective in the face of metabolic stressors. Based on this hypothesis; the first two major goals of the proposed studies are (1) to determine the effect of gain or loss of UCP function in transgenic mice on myocardial fatty acid metabolism and mitochondrial function and (2) to determine the effect of gain or loss of UCP function on left ventricular function and myocyte survival in the setting of ischemia/reperfusion or pressure overload hypertrophy. In addition to determining the functional consequences of changes in UCP expression, the proposed studies will characterize the regulation of UCP expression. Preliminary work from our lab has suggested that UCP2 expression is regulated by the metabolic stress kinase AMP-activated protein kinase (AMPK) while UCP3 expression is regulated by peroxisome proliferator activator receptor-alpha. Therefore, the third goal of the proposed studies is to elucidate the mechanism by which AMPK regulates UCP2 expression and to determine if there are differences in the regulation of UCP2 and UCP3 expression in response to metabolic stressors known to activate AMPK (e.g., ischemia, increased workload). It is anticipated that the results of these studies will yield important insights into the role of UCPs in the clinically important disorders of myocardial ischemia/reperfusion and heart failure.